Scaffold‐Free, Engineered Porcine Cartilage Construct for Cartilage Defect Repair—In Vitro and In Vivo Study

Abstract

This study introduces an implantable scaffold-free (SF) cartilage tissue construct that is composed of chondrocytes and their self-produced extracellular matrix (ECM). Chondrocytes were isolated from the articular cartilages from knees of domestic pigs (2-week old) and monolayer-cultured for 3-4 days in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 50 microg/mL of ascorbic acid. Briefly treated with 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA), an intact chondrocytes/ECM membrane, as a cell sheet was released from the plate bottom and subsequently centrifuged into a pellet-type construct. Each was grown in vitro for up to 5 weeks and subjected to various assays at different time points (1, 7, 14, 21, and 35 days). For in vivo implantation, full-thickness defects (n = 4) were manually created on the femoro-patellar groove of the left porcine knee and 1-week-cultured SF construct was implanted as an allograft for a month. One defect (#1) was an empty control and the remaining three received different recipes; construct only (#2) or 0.25% trypsin/EDTA-treated first and then construct and collagen gel (#3) or construct and collagen gel (#4). While the total cell numbers significantly increased by 2 weeks and then remained stable, cell viability stayed in the mid-70% range through the entire culture period. Biochemical assay found continuous glycosaminoglycan (GAG) accumulation. Histology exhibited that cell distribution was even in the construct and GAG intensity became stronger and uniform with time. Real-time reverse transcription polymerase chain reaction (RT-PCR) results showed that phenotypic stability peaked at 2 weeks, which was arable to that of freshly isolated chondrocytes. Upon analysis of the retrieved implants, some promising results were witnessed in the defects (#3) retaining not only their intact mass but also chondrocytic morphology with lacuna formation.

Scaffold-Free Cartilage Fabrication System Using Passaged Porcine Chondrocytes and Basic Fibroblast Growth Factor

“…We have developed previously a novel scaffold-free cartilage tissue formation system using a sequential process of high-density primary cultured cells and subsequent threedimensional (3D) pellet culture. 12 The cartilage tissue fabricated by the novel scaffold-free system showed satisfactory results in terms of the construct size, histological observation, and mechanical strength, which are significantly superior to traditional single-step pellet culture protocol. However, autologous chondrocytes from small biopsy of cartilage tissue are limited in cell number and inevitably have to be expanded through multiple passages, which cause dedifferentiation or loss of chondrogenic function of cells.…”

“…12 Cells were resuspended in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum, 100 U=mL penicillin G, and 100 mg=mL streptomycin. Cells were cultured in 150 mm dishes at 1Â10 4 cells=cm 2 in a humidified incubator at 378C under 5% CO 2 .…”

“…The scaffold-free cartilage was fabricated basically as described previously 12 except that passaged chondrocytes were used instead of primary cells and bFGF was treated during the culture. Briefly, the scaffold-free system was prepared via three steps: (i) the expansion of primary chondrocytes for two passages as described above, (ii) the high-density cell culture (2Â10 5 cells=cm 2 ) for 5-7 days to form cell sheets, and (iii) the pellet culture to fabricate a new cartilaginous tissue 1, 7, or 21 days.…”

Scaffold-free Cartilage Tissue by Mechanical Stress Loading for Tissue Engineering

“…But the phenotype control of chondrocytes in the scaffold was not enough. On the other hand, scaffold-free cartilage tissues formed by methods using alginate gel (Matsuda, 2003;Stoddart et al, 2006a), agarose gel (Kelm et al, 2004), rotational wall vessel (Marlovits, 2003), special system (Maini-Varlet, 2001;Grogan, 2003;Wang, 2004;Park, 2006), and a suspension culture of aggregating chondrocytes (Furukawa, 2003;Tsuchiya, 2005) are reported, and the phenotype of chondrocytes in the scaffold-free cartilage was possible to control , but these methods could not freely form cartilage tissues into arbitrary shapes. Furukawa et al (2008) succeeded in the formation of cartilage tissue into arbitrary shapes using two kinds of techniques.…”

Sequential Use of Fibroblastic Growth Factor 2 and Transforming Growth Factor β1 in Cartilage Tissue Engineering Using Canine Chondrocytes

“…However, many growth factors may produce dramatically different results. More specifically, Transforming Growth Factor 1 (TGF 1) and Fibroblastic Growth Factor 2 (FGF2) have produced variable results depending on the culture conditions, species and type of cell used [8][9][10][11][12][13][14][15][16][17][18][19]. Additionally, the biosynthetic activity may be influenced by sequential or concomitant use of growth factors [17].…”